Basic and Translational Science Enhanced Antitumor Efficacy of Integrin-targeted Oncolytic Adenovirus AxdAdB3-F/RGD on Bladder Cancer Hua Wang, Zhijian Cai, Fei Yang, Juan Luo, Makoto Satoh, Yoichi Arai, and Dechuan Li OBJECTIVE METHODS
RESULTS
CONCLUSION
To evaluate the therapeutic efficacy of AxdAdB-3 with Arg-Gly-Asp (RGD)-fiber modification (AxdAdB3-F/RGD), which enables integrin-dependent infection in bladder cancers. Flow cytometric analysis was applied to evaluated adenovirus-mediated gene transduction into various cells. The cytopathic effects of AxdAdB3-F/RGD were evaluated in bladder cancer cell lines and a normal bladder mucosa-derived cell line (HCV29) with AxCAZ3-F/RGD (control) or AxdAdB-3. The efficacy of bladder instillation therapy with AxdAdB3-F/RGD for orthotopic bladder cancer was investigated in nude mice. Expression of coxsackievirus adenovirus receptor (CAR) and integrins (avb3 and avb5) vary in different bladder cancer cell lines. The susceptibility of various cell lines to adenovirus was associated with the expression of CAR. AxdAdB-3 was more cytopathic in CAR-positive bladder cancer cells than in CAR-negative cells, whereas AxdAdB3-F/RGD caused effective oncolysis in both CAR-positive and CAR-negative bladder cancer cells. AxdAdB3-F/RGD was not cytotoxic to HCV29 cells. Direct instillation of AxdAdB3-F/RGD into the bladder of the orthotopic model, established by CAR-deficient human bladder cancer cells, inhibited tumor growth and led to significantly elongated survival. E1A and E1B double-restricted oncolytic adenovirus with RGD fiber modification has enhanced infectivity and oncolytic effects to CAR-deficient bladder cancers, suggesting the therapeutic potential of AxdAdB3-F/RGD for bladder cancers. UROLOGY 83: 508.e13e508.e19, 2014. 2014 Elsevier Inc.
B
ladder cancer is a common malignancy, with an estimated 73,510 newly diagnosed cases and 14,880 deaths from this disease in the United States in 2012.1 More than 70% of patients with bladder cancers have nonmuscle-invasive disease, and approximately 25% of patients initially present with muscle invasion. Transurethral resection of the bladder tumor, with or without intravesical instillation of bacille CalmetteGuerin or chemotherapeutic agents, is the current standard therapy for nonmuscle-invasive bladder cancer. However, approximately 20%-30% of patients have recurrent cancer after initial treatment, and 10%-20% will progress to muscle-invasive disease that requires more
H.W. and Z.C. contributed equally. Financial Disclosure: The authors declare that they have no relevant financial interests. Funding Support: This work was supported by grant 2009R10001 from the Qianjiang Program of Zhejiang Province, China. From the Department of Urology, Zhejiang Cancer Hospital, Hangzhou, China; the Institute of Immunology, School of Medicine, Zhejiang University, Hangzhou, China; the Department of Pathology, Zhejiang Cancer Hospital, Hangzhou, China; the Department of Urology, Sen-en General Hospital, Tagajo, Miyagi, Japan; and the Department of Urology, Tohoku University Graduate School of Medicine, Sendai, Japan Reprint requests: Hua Wang, M.D., Ph.D., Department of Urology, Zhejiang Cancer Hospital, 38 Guangji Road, Gongshu District, Hangzhou 310022, China. E-mail: [email protected] Submitted: March 19, 2013, accepted (with revisions): October 18, 2013
508.e13 ª 2014 Elsevier Inc. All Rights Reserved
aggressive therapies.2 Patients with muscle-invasive bladder cancer have a 50% risk of distant metastases, causing a poor prognosis.3 Therefore, there is an urgent need to develop new treatment modalities to prevent the recurrence and progression to muscle-invasive bladder cancer and improve the prognosis of bladder cancer. It has been reported that oncolytic adenovirus with deletion of E1B-55kD or mutated E1A is able to replicate selectively in, and kill, cancer cells that have deficient p53 or disrupted Rb pathway while sparing normal cells.4 Preclinical and clinical trials both have demonstrated that oncolytic adenovirus is a promising treatment for malignant tumors, including bladder cancer.4-6 We have previously demonstrated that the E1A, E1B double mutant oncolytic adenovirus AxdAdB-3 has a potential antitumor effect on an orthotopic bladder cancer model in severe combined immunodeficiency mice.7 However, AxdAdB-3 showed insufficient cytopathic effects in some bladder cancer lines that have low expression of coxsackievirus adenovirus receptor (CAR).7 Because CAR plays a crucial role in adenoviral infection, cancer cells with decreased expression of CAR are resistant to viral infection and, therefore, Ad-mediated gene therapy.8 Bladder cancer cell lines9 and tumors,10 especially those at an advanced stage and high grade, frequently lose 0090-4295/14/$36.00 http://dx.doi.org/10.1016/j.urology.2013.10.025
CAR expression, which limits the use of oncolytic adenovirus for treating bladder cancer. It has been reported that inserting the Arg-Gly-Asp (RGD) peptide into the HI loop of the fiber knob domain enhances adenovirus-mediated gene transduction to CAR-negative cells through the binding of the RGD peptide to integrins on the target cells.11 In the present study, we evaluated the therapeutic efficacy of the E1A, E1B double mutant oncolytic adenovirus AxdAdB-3 with RGD fiber modification (AxdAdB3-F/RGD) for bladder cancers.
MATERIALS AND METHODS Cell Lines YTS-112 (p53 mutant and p16 homozygous deletion7) and KK4713 (both p5314 and p16 wild type), both determined by loss of heterozygosity analysis and mutation analysis with direct sequencing, derived from human muscle-invasive and human nonmuscle-invasive bladder cancer, and HCV29, derived from human normal bladder mucosa, were provided by the Department of Urology, Tohoku University School of Medicine (Sendai, Japan). Human bladder cancer cell lines T24 and 5637 (p53 mutation and p16 wild type7) and human embryonic kidney 293 cells (HEK-293) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All 4 bladder cancer cell lines, with high expression of uroplakin, were determined by reverse transcriptase polymerase chain reaction, confirming the urothelial origin in these cell lines. The cell lines were maintained in Roswell Park Memorial Institute or Eagle’s minimal essential medium (for HEK293) supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 mg/ mL streptomycin in a humidified 5% CO2 atmosphere at 37 C.
Generation of Recombinant Adenovirus AxCAlacZ is a recombinant adenovirus lacking the E1 region,15 and AxdAdB-3 is a recombinant adenovirus with mutations of the 122nd and 124th amino acids in CR-2 of E1A, eliminating the ability for Rb binding and deficient in E1B55KD.4 AxCAZ3-F/RGD and AxdAdB3-F/RGD were constructed by inserting the RGD peptide at the HI loop of the fiber knob domain in AxCAlacZ and AxdAdB-3, respectively.16 The viruses were provided by the RIKEN Gene Bank (Tsukuba, Japan). All adenoviruses were packaged in HEK-293 cells, and the titer was determined by standard plaque assays.
Determination of CAR and Integrins The expression of CAR and integrins (avb3 and avb5) in the
cell lines was measured by flow cytometry. In brief, cells were detached by an enzyme-free dissociation solution and were incubated on ice for 1 hour with the primary antibody RmcB (mouse antihuman CAR monoclonal antibody, Upstate Biotechnology, Lake Placid, NY), sc-52684 (a mouse antihuman monoclonal antibody against integrin avb3, Santa Cruz Biotechnology, Santa Cruz, CA), and P5H9 (a mouse antihuman monoclonal antibody against Integrin avb5, R&D Systems, Inc., Minneapolis, MN). After several washes, the cells were incubated with fluorescein isothiocyanate-conjugated secondary antibody for 30 minutes. The cells were immediately analyzed by flow cytometry. CAR expression was also determined by western blot analysis. In brief, cells were lysed in 100 mL of lysis buffer. Equal amounts of protein in the supernatant UROLOGY 83 (2), 2014
were run on SDS-PAGE (12% gel). Blots were probed by antiCAR antibody, RmcB.
Infectivity of Adenoviruses Cells were infected with AxCAlacZ or AxCAZ3-F/RGD at a multiplicity of infection of 100 for 24 hours. The expression of adenoviral hexon protein in infected cells was determined using flow cytometric analysis (as described previously) with antihexon mouse monoclonal antibody (MAB805, Chemicon International, Temecula, CA).
Cytopathic Effects of Oncolytic Adenovirus Cells (3 103 per well) were seeded in 96-well plates and infected with AxCAlacZ, AxCAZ3-F/RGD, AxdAdB-3, or AxdAdB3-F/RGD at a multiplicity of infection of 30. Viable cells and cytopathic effects were evaluated by cell counting kit-8 (CCK-8, Dojindo, Japan) according to the manufacturer’s protocol. Briefly, 10 mL of CCK-8 solution was added to each well, and the cells were incubated for 4 hours. The absorbance of the colored formazan was measured using a microplate reader at 450 nm. To translate the assay results to the numbers of viable cells, standard curves were made in each experiment.
Animal Studies An orthotopic bladder cancer model was established in nude mice, and viruses were administered intravesically by a transurethral catheter as described.7,17 In brief, 8-week-old, female, BALB/C mice were anesthetized with phenobarbital sodium (75 mg/kg intraperitoneally). The bladder was catheterized with a 24-gauge intravenous catheter, and 100 mL of 0.2% trypsin was infused and retained in the bladder for 10 minutes. After the bladder was washed with phosphate-buffered saline (PBS), YTS1 cells (1 106) in 100 mL of PBS were instilled into the bladder. Three days later, the bladder was recatheterized and instilled with 1 108 plaque-forming units of viruses in 100 mL. We measured survival by treating mice with bladder tumors with AxCAZ3-F/RGD, AxdAdB-3, or AxdAdB3-F/RGD (1 108 plaque-forming units) with a single intravesical instillation on day 3 after tumor inoculation. Adenoviral hexon protein was analyzed by immunohistochemistry to determine adenoviral replication within the bladder tumors. Paraffin-embedded sections were deparaffinized, rehydrated with xylol and ethanol in PBS, and then immunostained according to the manufacturer’s instructions using labeled streptavidin biotin (LSABkit, Dako, Japan) with antihexon mouse monoclonal antibody (MAB805, Chemicon International, Temecula, CA).7
Statistical Analysis Statistical analysis was performed using SPSS for Windows version 19 software system (SPSS Inc., Chicago). The results were presented as the mean standard deviation. The results other than those of survival were statistically analyzed using an unpaired t test. Survival was assessed by the Kaplan-Meier method, and the log-rank test was used to determine the differences between the treatment group and control. P
RESULTS
CONCLUSION
To evaluate the therapeutic efficacy of AxdAdB-3 with Arg-Gly-Asp (RGD)-fiber modification (AxdAdB3-F/RGD), which enables integrin-dependent infection in bladder cancers. Flow cytometric analysis was applied to evaluated adenovirus-mediated gene transduction into various cells. The cytopathic effects of AxdAdB3-F/RGD were evaluated in bladder cancer cell lines and a normal bladder mucosa-derived cell line (HCV29) with AxCAZ3-F/RGD (control) or AxdAdB-3. The efficacy of bladder instillation therapy with AxdAdB3-F/RGD for orthotopic bladder cancer was investigated in nude mice. Expression of coxsackievirus adenovirus receptor (CAR) and integrins (avb3 and avb5) vary in different bladder cancer cell lines. The susceptibility of various cell lines to adenovirus was associated with the expression of CAR. AxdAdB-3 was more cytopathic in CAR-positive bladder cancer cells than in CAR-negative cells, whereas AxdAdB3-F/RGD caused effective oncolysis in both CAR-positive and CAR-negative bladder cancer cells. AxdAdB3-F/RGD was not cytotoxic to HCV29 cells. Direct instillation of AxdAdB3-F/RGD into the bladder of the orthotopic model, established by CAR-deficient human bladder cancer cells, inhibited tumor growth and led to significantly elongated survival. E1A and E1B double-restricted oncolytic adenovirus with RGD fiber modification has enhanced infectivity and oncolytic effects to CAR-deficient bladder cancers, suggesting the therapeutic potential of AxdAdB3-F/RGD for bladder cancers. UROLOGY 83: 508.e13e508.e19, 2014. 2014 Elsevier Inc.
B
ladder cancer is a common malignancy, with an estimated 73,510 newly diagnosed cases and 14,880 deaths from this disease in the United States in 2012.1 More than 70% of patients with bladder cancers have nonmuscle-invasive disease, and approximately 25% of patients initially present with muscle invasion. Transurethral resection of the bladder tumor, with or without intravesical instillation of bacille CalmetteGuerin or chemotherapeutic agents, is the current standard therapy for nonmuscle-invasive bladder cancer. However, approximately 20%-30% of patients have recurrent cancer after initial treatment, and 10%-20% will progress to muscle-invasive disease that requires more
H.W. and Z.C. contributed equally. Financial Disclosure: The authors declare that they have no relevant financial interests. Funding Support: This work was supported by grant 2009R10001 from the Qianjiang Program of Zhejiang Province, China. From the Department of Urology, Zhejiang Cancer Hospital, Hangzhou, China; the Institute of Immunology, School of Medicine, Zhejiang University, Hangzhou, China; the Department of Pathology, Zhejiang Cancer Hospital, Hangzhou, China; the Department of Urology, Sen-en General Hospital, Tagajo, Miyagi, Japan; and the Department of Urology, Tohoku University Graduate School of Medicine, Sendai, Japan Reprint requests: Hua Wang, M.D., Ph.D., Department of Urology, Zhejiang Cancer Hospital, 38 Guangji Road, Gongshu District, Hangzhou 310022, China. E-mail: [email protected] Submitted: March 19, 2013, accepted (with revisions): October 18, 2013
508.e13 ª 2014 Elsevier Inc. All Rights Reserved
aggressive therapies.2 Patients with muscle-invasive bladder cancer have a 50% risk of distant metastases, causing a poor prognosis.3 Therefore, there is an urgent need to develop new treatment modalities to prevent the recurrence and progression to muscle-invasive bladder cancer and improve the prognosis of bladder cancer. It has been reported that oncolytic adenovirus with deletion of E1B-55kD or mutated E1A is able to replicate selectively in, and kill, cancer cells that have deficient p53 or disrupted Rb pathway while sparing normal cells.4 Preclinical and clinical trials both have demonstrated that oncolytic adenovirus is a promising treatment for malignant tumors, including bladder cancer.4-6 We have previously demonstrated that the E1A, E1B double mutant oncolytic adenovirus AxdAdB-3 has a potential antitumor effect on an orthotopic bladder cancer model in severe combined immunodeficiency mice.7 However, AxdAdB-3 showed insufficient cytopathic effects in some bladder cancer lines that have low expression of coxsackievirus adenovirus receptor (CAR).7 Because CAR plays a crucial role in adenoviral infection, cancer cells with decreased expression of CAR are resistant to viral infection and, therefore, Ad-mediated gene therapy.8 Bladder cancer cell lines9 and tumors,10 especially those at an advanced stage and high grade, frequently lose 0090-4295/14/$36.00 http://dx.doi.org/10.1016/j.urology.2013.10.025
CAR expression, which limits the use of oncolytic adenovirus for treating bladder cancer. It has been reported that inserting the Arg-Gly-Asp (RGD) peptide into the HI loop of the fiber knob domain enhances adenovirus-mediated gene transduction to CAR-negative cells through the binding of the RGD peptide to integrins on the target cells.11 In the present study, we evaluated the therapeutic efficacy of the E1A, E1B double mutant oncolytic adenovirus AxdAdB-3 with RGD fiber modification (AxdAdB3-F/RGD) for bladder cancers.
MATERIALS AND METHODS Cell Lines YTS-112 (p53 mutant and p16 homozygous deletion7) and KK4713 (both p5314 and p16 wild type), both determined by loss of heterozygosity analysis and mutation analysis with direct sequencing, derived from human muscle-invasive and human nonmuscle-invasive bladder cancer, and HCV29, derived from human normal bladder mucosa, were provided by the Department of Urology, Tohoku University School of Medicine (Sendai, Japan). Human bladder cancer cell lines T24 and 5637 (p53 mutation and p16 wild type7) and human embryonic kidney 293 cells (HEK-293) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All 4 bladder cancer cell lines, with high expression of uroplakin, were determined by reverse transcriptase polymerase chain reaction, confirming the urothelial origin in these cell lines. The cell lines were maintained in Roswell Park Memorial Institute or Eagle’s minimal essential medium (for HEK293) supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 mg/ mL streptomycin in a humidified 5% CO2 atmosphere at 37 C.
Generation of Recombinant Adenovirus AxCAlacZ is a recombinant adenovirus lacking the E1 region,15 and AxdAdB-3 is a recombinant adenovirus with mutations of the 122nd and 124th amino acids in CR-2 of E1A, eliminating the ability for Rb binding and deficient in E1B55KD.4 AxCAZ3-F/RGD and AxdAdB3-F/RGD were constructed by inserting the RGD peptide at the HI loop of the fiber knob domain in AxCAlacZ and AxdAdB-3, respectively.16 The viruses were provided by the RIKEN Gene Bank (Tsukuba, Japan). All adenoviruses were packaged in HEK-293 cells, and the titer was determined by standard plaque assays.
Determination of CAR and Integrins The expression of CAR and integrins (avb3 and avb5) in the
cell lines was measured by flow cytometry. In brief, cells were detached by an enzyme-free dissociation solution and were incubated on ice for 1 hour with the primary antibody RmcB (mouse antihuman CAR monoclonal antibody, Upstate Biotechnology, Lake Placid, NY), sc-52684 (a mouse antihuman monoclonal antibody against integrin avb3, Santa Cruz Biotechnology, Santa Cruz, CA), and P5H9 (a mouse antihuman monoclonal antibody against Integrin avb5, R&D Systems, Inc., Minneapolis, MN). After several washes, the cells were incubated with fluorescein isothiocyanate-conjugated secondary antibody for 30 minutes. The cells were immediately analyzed by flow cytometry. CAR expression was also determined by western blot analysis. In brief, cells were lysed in 100 mL of lysis buffer. Equal amounts of protein in the supernatant UROLOGY 83 (2), 2014
were run on SDS-PAGE (12% gel). Blots were probed by antiCAR antibody, RmcB.
Infectivity of Adenoviruses Cells were infected with AxCAlacZ or AxCAZ3-F/RGD at a multiplicity of infection of 100 for 24 hours. The expression of adenoviral hexon protein in infected cells was determined using flow cytometric analysis (as described previously) with antihexon mouse monoclonal antibody (MAB805, Chemicon International, Temecula, CA).
Cytopathic Effects of Oncolytic Adenovirus Cells (3 103 per well) were seeded in 96-well plates and infected with AxCAlacZ, AxCAZ3-F/RGD, AxdAdB-3, or AxdAdB3-F/RGD at a multiplicity of infection of 30. Viable cells and cytopathic effects were evaluated by cell counting kit-8 (CCK-8, Dojindo, Japan) according to the manufacturer’s protocol. Briefly, 10 mL of CCK-8 solution was added to each well, and the cells were incubated for 4 hours. The absorbance of the colored formazan was measured using a microplate reader at 450 nm. To translate the assay results to the numbers of viable cells, standard curves were made in each experiment.
Animal Studies An orthotopic bladder cancer model was established in nude mice, and viruses were administered intravesically by a transurethral catheter as described.7,17 In brief, 8-week-old, female, BALB/C mice were anesthetized with phenobarbital sodium (75 mg/kg intraperitoneally). The bladder was catheterized with a 24-gauge intravenous catheter, and 100 mL of 0.2% trypsin was infused and retained in the bladder for 10 minutes. After the bladder was washed with phosphate-buffered saline (PBS), YTS1 cells (1 106) in 100 mL of PBS were instilled into the bladder. Three days later, the bladder was recatheterized and instilled with 1 108 plaque-forming units of viruses in 100 mL. We measured survival by treating mice with bladder tumors with AxCAZ3-F/RGD, AxdAdB-3, or AxdAdB3-F/RGD (1 108 plaque-forming units) with a single intravesical instillation on day 3 after tumor inoculation. Adenoviral hexon protein was analyzed by immunohistochemistry to determine adenoviral replication within the bladder tumors. Paraffin-embedded sections were deparaffinized, rehydrated with xylol and ethanol in PBS, and then immunostained according to the manufacturer’s instructions using labeled streptavidin biotin (LSABkit, Dako, Japan) with antihexon mouse monoclonal antibody (MAB805, Chemicon International, Temecula, CA).7
Statistical Analysis Statistical analysis was performed using SPSS for Windows version 19 software system (SPSS Inc., Chicago). The results were presented as the mean standard deviation. The results other than those of survival were statistically analyzed using an unpaired t test. Survival was assessed by the Kaplan-Meier method, and the log-rank test was used to determine the differences between the treatment group and control. P
